Project description:Transient siRNA-mediated knockdown of hnRNP L, followed by cycloheximide treatment to eliminate NMD. Supplementary files GSE37561_activated_exon.txt and GSE37561_repressed_exon.txt list exons predicted to be activated and repressed by hnRNPL, respectively. 3 biological replicates of hnRNP L (luciferase) knockdown samples
Project description:One day before transfection, HeLa cells were seeded in 6-well culture plates (1.5 x 10e5 cells per well) or 10-cm culture dishes (4.3 x 10e5 cells per dish). siRNA duplex (at a final concentration in culture medium of 30 nM) was transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. siRNA duplices specific for human hnRNP L, human hnRNP LL, and luciferase GL2 were from MWG Biotech (Ebersberg, Germany). Keywords: control / knockdown comparison
Project description:Transient siRNA-mediated knockdown of hnRNP L, followed by cycloheximide treatment to eliminate NMD. Supplementary files GSE37561_activated_exon.txt and GSE37561_repressed_exon.txt list exons predicted to be activated and repressed by hnRNPL, respectively.
Project description:We performed EGF treatment and hnRNP A1 knockdown in HeLa cells and analyzed alternative splicing patterns by high-throughput RNA sequencing.
Project description:poly(A)+ RNA samples from hnRNP C siRNA knockdown and control HeLa cells were compared on a splice-junction microarray to detect changes in alternative splicing. Using the ASPIRE3 algorithm, we detected changes in splicing at 1,340 alternative exons. We observed a similar incidence of increased or decreased exon inclusion in hnRNP C knockdown cells, indicating that hnRNP C can either silence or enhance exon inclusion, respectively. We validated changes at 26 exons by RT-PCR with a 92% success rate.
Project description:The nuclear matrix associated hnRNP U/SAF-A protein has been implicated in diverse pathways from transcriptional regulation to telomere length control to X inactivation, but the precise mechanism underlying each of these processes has remained elusive. Here, we report hnRNP U as a regulator of SMN2 splicing from a custom RNAi screen. Genome-wide analysis by CLIP-seq reveals that hnRNP U binds virtually to all classes of regulatory non-coding RNAs, including all snRNAs required for splicing of both major and minor classes of introns, leading to the discovery that hnRNP U regulates U2 snRNP maturation and Cajal body morphology in the nucleus. Global analysis of hnRNP U-dependent splicing by RNA-seq coupled with bioinformatic analysis of associated splicing signals suggests a general rule for splice site selection through modulating the core splicing machinery. These findings exemplify hnRNP U/SAF-A as a potent regulator of nuclear ribonucleoprotein particles in diverse gene expression pathways. Examination of hnRNP U regulated splicing in Hela cells with CLIP-seq (two biological replicates) and paired-end RNA-seq (control and hnRNP U knockdown)
Project description:iCLIP experiment to assess the binding of the highly abundant nuclear RNA-binding protein hnRNP C and core splicing factor U2AF65 on a genomic scale. To investigate how both proteins compete for binding at a subset of sites, U2AF65 iCLIP experiments were performed from both HNRNPC knockdown and control HeLa cells.
Project description:Expression profiling of fetal liver erythroid precursors after either Hipk1 or Hipk2 knockdown by shRNA versus control shRNA Two condition experiment, Hipk1 or Hipk2 knockdown versus control (shRNA against luciferase), two replicates each shRNA
Project description:In order to measure the impact of Rpb9 on co-transcriptional splicing, we treated HeLa cells with control siRNA or siRpb9, and for comparative analysis, we also depleted Rpb1 and confirmed by Western blotting the knockdown efficiency in all cases. To measure co-transcriptional splicing, we fractionated HeLa cells into cytosolic (Cyt), nucleoplasm (NP), and chromatin/particle (Chr) fractions. We purified total RNA from each compartment, spiked in with in vitro transcribed EGFP and FireFly luciferase RNA for data normalization, depleted ribosomal RNAs, and constructed two independent libraries for RNA-seq, which showed excellent reproducibility .
Project description:Individual-nucleotide resolution UV-crosslinking and immunoprecipitation (iCLIP) combined with high-throughput sequencing was used to generate a transcriptome-wide binding map of hnRNP L. Supplementary file GSE37560_hnRNPL_crosslink_site.bed includes filtered crosslink sites of hnRNPL: combining data from all 3 experiments. 3 biological replicates of hnRNP L-specific and control (Flag) co-immunoprecipitated RNA after UV-crosslinking in HeLa cells
